The Top 10 oomycete pathogens in molecular plant pathology

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The Top 10 oomycete pathogens in molecular plant pathology 1 1

Sophien Kamoun1*, Oliver Furzer1, Jonathan D.G. Jones1, Howard S. Judelson2, Gul Shad Ali3, Ronaldo J. 2 D. Dalio4, Sanjoy Guha Roy5, Leonardo Schena6, Antonios Zambounis7, Franck Panabières8, David Cahill9, 3 Michelina Ruocco10, Andreia Figueiredo11, Xiao-Ren Chen12, Jon Hulvey13, Remco Stam14, Kurt Lamour15, 4 Mark Gijzen16 Brett M. Tyler17, Niklaus J. Grünwald18, M. Shahid Mukhtar19, 20, Daniel F. A. Tomé21, Mahmut 5 Tör22, Guido Van den Ackerveken23, John McDowell24, Fouad Daayf25, William E. Fry26, Hannele Lindqvist-6 Kreuze27, Harold J.G. Meijer28, Benjamin Petre1,29, Jean Ristaino30, Kentaro Yoshida1, Paul R.J. Birch14, and 7 Francine Govers28 8

1The Sainsbury Laboratory, Norwich Research Park, Norwich, NR4 7UH, UK. 9 2Department of Plant Pathology and Microbiology, University of California, Riverside, CA 92521 USA 10 3Department of Plant Pathology and MREC, IFAS, University of Florida, Apopka, FL 32703, USA 11 4Biotechnology lab, Centro de Citricultura Sylvio Moreira / Instituto Agronomico, Cordeirópolis-Sao Paulo, 12 Brazil 13 5Department of Botany, West Bengal State University, Barasat, Kolkata-700126, India 14 6Dipartimento di Gestione dei Sistemi Agrari e Forestali, Università degli Studi Mediterranea, 89122 Reggio 15 Calabria, Italy 16 7UMR1290 BIOGER-CPP, INRA-AgroParisTech, 78850 Thiverval-Grignon, France 17 8INRA, UMR1355, Univ. Nice Sophia Antipolis, CNRS, UMR 7254, ISA, F-06903 Sophia Antipolis, France 18 9Deakin University, Geelong, Victoria, 3217, Australia 19 10Portici Division of The Italian National Research Council (CNR) Institute for Sustainable Plant Protection 20 (IPSP) Via Università 133, 80055 Portici (NA) Italy 21 11Centre for Biodiversity, Functional and Integrative Genomics, Faculty of Sciences, University of Lisboa, 22 1749-016 Lisboa, Portugal. 23 12College of Horticulture and Plant Protection, Yangzhou University, China 24 13Stockbridge School of Agriculture, University of Massachusetts Amherst, USA 25 14Division of Plant Sciences, College of Life Sciences, University of Dundee (at James Hutton Institute), 26 Errol Road, Invergowrie DD2 5DA, UK 27 15Department of Entomology and Plant Pathology, University of Tennessee, USA 28 16Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario N5V 4T3, Canada 29 17Center for Genome Research and Biocomputing, and Department of Botany and Plant Pathology, Oregon 30 State University, Corvallis, OR 97331, USA. 31 18USDA ARS, Plant Pathology Horticultural Crops Research Lab. 3420 NW Orchard Ave., Corvallis, Oregon 32 97330, United States 33 19Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA 34 20Nutrition Obesity Research Center, University of Alabama at Birmingham, Birmingham, 35294, AL, USA 35 21School of Life Sciences, University of Warwick, Coventry, CV4 7AL, UK 36 22National Pollen and Aerobiology Research Unit, The University of Worcester, Henwick Grove, Worcester 37 WR2 6 AJ, UK 38 23Plant- Microbe Interactions, Department of Biology, Utrecht University, Padualaan 8, 3584 CH Utrecht, 39 Netherlands 40 24Department of Plant Pathology, Physiology and Weed Science, Virginia Tech, Blacksburg, VA 24061, USA 41 25Department of Plant Science, University of Manitoba, Winnipeg, MB, Canada 42 26Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, New York 14853, 43 USA 44

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27International Potato Center, Apartado 1558, Lima 12, Peru 45 28Laboratory of Phytopathology, Wageningen University, NL-1-6708 PB Wageningen, Netherlands 46 29INRA, UMR 1136 Interactions Arbres/Microorganismes, Centre INRA Nancy Lorraine, 54280 47 Champenoux, France 48 30Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina 27695, USA 49 50 * Corresponding author E-mail: [email protected] 51

Running header: Top 10 oomycete plant pathogens 52 53 SUMMARY 54 55 Oomycetes form a deep lineage of eukaryotic organisms that includes a large number of plant pathogens 56 that threaten natural and managed ecosystems. We undertook a survey to query the community for their 57 ranking of plant pathogenic oomycete species based on scientific and economic importance. In total, we 58 received 263 votes from 62 scientists in 15 countries for a total of 33 species. The Top 10 species and their 59 ranking are: (1) Phytophthora infestans; (2, tied) Hyaloperonospora arabidopsidis; (2, tied) Phytophthora 60 ramorum; (4) Phytophthora sojae; (5) Phytophthora capsici; (6) Plasmopara viticola; (7) Phytophthora 61 cinnamomi; (8, tied) Phytophthora parasitica; (8, tied) Pythium ultimum; and (10) Albugo candida. The article 62 provides an introduction to these 10 taxa and a snapshot of current research. We hope that the list will serve 63 as a benchmark for future trends in oomycete research. 64 65 66 INTRODUCTION 67 68 Oomycetes are eukaryotic organisms that superficially resemble filamentous fungi but are phylogenetically 69 related to diatoms and brown algae in the stramenopiles (Gunderson et al., 1987; Jiang and Tyler, 2012; 70 Lamour and Kamoun, 2009; Thines, 2014; Thines and Kamoun, 2010). Fossil evidence indicates that a 71 number of oomycetes emerged as endophytes of land plants at least by the Carboniferous period ~300-350 72 million years ago (Krings et al., 2011). One species, Combresomyces williamsonii, described from ~320 73 million year-old petrified stem cortex and rootlets of a seed fern may have even been parasitic (Strullu-74 Derrien et al., 2011). Phylogenetic analyses of modern taxa revealed that plant parasitism has evolved 75 independently in three lineages of oomycetes (Thines and Kamoun, 2010). Well known plant pathogens, 76 namely downy mildews, Phytophthora and Pythium, appear to have radiated from a common plant parasitic 77 ancestor (Thines and Kamoun, 2010). The impact of oomycetes on mankind is well documented both as 78 persistent threats to subsistence and commercial farming, and as destructive pathogens of native plants 79 (Agrios, 2005; Erwin and Ribeiro, 1996; Lamour and Kamoun, 2009). As a result, news related to plant 80 diseases caused by oomycetes tends to capture the interest of the general public and is frequently featured 81 in the media. 82

In the last two decades, increased awareness of the distinctive phylogeny and biology of 83 oomycetes drove the emergence of a specialist research community that is currently organized under the 84 umbrella of the “Oomycete Molecular Genetics Network”. This community has moved the field beyond the 85 gloomy view of the 1980s that oomycetes are a ‘fungal geneticist’s nightmare’ (Shaw, 1983; discussed in 86 Schornack et al, 2009). It produced novel paradigms in understanding host-microbe interactions, effector 87 biology and genome evolution (Bozkurt et al., 2012; Govers and Gijzen, 2006; Jiang and Tyler, 2012; 88 Schornack et al., 2009; Vleeshouwers et al., 2011). The oomycete community was one of the first in plant 89 pathology to initiate coordinated transcriptome and genome sequencing projects, and subsequently to 90 exploit the resulting resources to drive conceptual advances (Schornack et al., 2009; Govers and Gijzen, 91 2006; Pais et al., 2013). These days, with the genomes serving as unique resources for basic and applied 92 research, oomycetes are best portrayed as a ‘genomicist’s dream’ (Jiang and Tyler, 2012; Schornack et al., 93 2009; Govers and Gijzen, 2006; Pais et al., 2013). 94 A

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It is, therefore, particularly fitting that the oomycetes are at last covered by the Top 10 review 95 series of Molecular Plant Pathology. The process to generate the list and the aim of this article are similar to 96 previous contributions on plant pathogenic viruses (Scholthof et al., 2011), fungi (Dean et al., 2012), 97 bacteria (Mansfield et al., 2012) and nematodes (Jones et al., 2013). We undertook a survey to query the 98 community for their ranking of plant pathogenic oomycete taxa based on scientific and economic 99 importance. In total, we received 263 votes from 62 scientists in 15 countries that yielded the Top 10 100 species (Table 1). To some degree the results reflect the sizes of the sub-communities. Six of the ten 101 species belong to the genus Phytophthora, which is more commonly studied than any other oomycete 102 genus. Obligate parasites are also well represented with three species (Hyaloperonospora arabidopsidis, 103 Plasmopara viticola, and Albugo candida, Table 1). Another 23 species received votes but ranked outside 104 the Top 10 (Table 2). The fish parasite Saprolegnia parasitica received enough votes to place it in the Top 105 10 but was removed given that the list focuses on plant pathogens (Table 2). 106 The article is based on contributions from the voters to provide an introduction to the 10 taxa and a 107 snapshot of current research. Each section starts with a brief overview of the selected species, e.g. its 108 importance, pathology, host range, and life cycle. This is followed by a review of current research themes, 109 particularly the unique findings that emerged from studying the particular species, and an outlook on future 110 research. We hope that this article will serve as a reference and resource for novice and experienced 111 readers alike, as well as provide a benchmark for future trends in oomycete research. 112 113 114 1. PHYTOPHTHORA INFESTANS 115 116 Phytophthora infestans causes potato late blight (Fig. 1), a disease with major historical impact. Providing 117 twice the calories of rye and wheat per hectare, potato was central to European agriculture between 1750 118 and 1850. In 1844 the arrival of P. infestans changed this situation. In addition to the well-documented Irish 119 famine (Fig. 2), crop failures in 1845 and 1846 contributed to an estimated 750,000 hunger-associated 120 deaths in Continental Europe (Zadoks, 2008). The discovery that late blight was caused by a microbial 121 pathogen, fifteen years before Pasteur’s formal confirmation of Germ Theory, places the 19th century 122 migration of P. infestans as a significant milestone in the foundation of plant pathology as a scientific 123 discipline. Today, late blight remains a major constraint to the production of potato, the world’s third largest 124 staple crop, and is thus a constant threat to food security (Fisher et al., 2012; Haverkort et al., 2008). 125 In the 1840s, trade in potatoes likely facilitated long-distance migration of the pathogen. Whole-126 genome sequencing of European herbarium samples has revealed populations belonging to the HERB-1 127 genotype that were different from US-1, the genotype dominating the population globally in the mid-to-late 128 20th century (Goodwin et al., 1994; Martin et al., 2013; Martin et al., 2014; Yoshida et al., 2013; Yoshida et 129 al., 2014). Waves of migration and genotype displacements in the 20th century have been well documented. 130 The introduction of the A2 mating type to Europe in the 1970s (Drenth et al., 1994), and more recently, the 131 emergence of new, more aggressive lineages such as 13_A2 (Blue 13) in Europe (Cooke et al., 2012), US-132 22 in Eastern United States (Fry et al., 2013; Hu et al., 2012), and US-23/US-24 in Canada (Peters et al., 133 2014) impose constant challenges to disease resistance breeding. 134

In the 1990s, P. infestans research led the development of molecular approaches to study 135 oomycete pathology and biology. The first DNA transformation system (Judelson et al., 1991) and the first 136 transcriptional profiling during infection (Pieterse et al., 1991) paved the way to detailed molecular 137 investigations of sexual and asexual development and pathogenicity (reviewed in Judelson et al., 1997). 138 DNA fingerprinting revolutionised population studies (Goodwin et al., 1992) and was a prelude to the first 139 genome-wide genetic map of a Phytophthora species (van der Lee et al., 1997). Observations that gene 140 silencing occurred in P. infestans transformants (e.g. Judelson and Whittacker, 1995) sparked important 141 studies of the mechanisms underlying transcriptional silencing (van West et al., 1999). The first large-scale 142 transcript sequencing studies (Kamoun et al., 1999) ushered in the genomics era of Phytophthora research. 143

In the last 15 years, advanced genomic and functional approaches put P. infestans at the forefront 144 of research to understand oomycete development (Judelson and Blanco, 2005) and pathogenicity (Kamoun, 145 A

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2006). An elegant combination of bioinformatics, proteomics and functional genomics revealed secreted 146 proteins as potential factors influencing host-P. infestans interactions (Torto et al., 2003). Apoplastic 147 effectors that inhibit host secreted proteases were first described in P. infestans (Tian et al., 2004) and the 148 apoplastic phytotoxin-like SCR74 (Liu et al., 2005) provided support for the use of diversifying selection as a 149 criterion for identifying candidate effectors. The apoplastic effectors EPIC1 and EPIC2B were found to 150 converge on the host protease RCR3 that is also targeted by an independently evolved effector from a 151 fungal plant pathogen (Song et al., 2009). Recently, diversifying selection of the EPIC1 protease inhibitor 152 was implicated in functional adaptation to the equivalent protease target after ‘jumps’ to a new host (Dong et 153 al., 2014). 154

The first reported P. infestans avirulence effector was AVR3a, the counterpart of resistance protein 155 R3a (Armstrong et al., 2005). Comparison with avirulence effectors from Phytophthora sojae and H. 156 arabidopsidis identified conserved amino acid motifs RXLR and EER (Rehmany et al., 2005), which act as 157 signals for translocation into host cells (Whisson et al., 2007). The genome sequence of P. infestans 158 revealed hundreds of genes predicted to encode RXLR effectors plus a second class of candidate effectors 159 called Crinklers (CRNs) (Haas et al., 2009), which may also be delivered inside plant cells (Schornack et al., 160 2010). These effector genes are rapidly evolving and are typically variable between different P. infestans 161 isolates (Cooke et al., 2012). RXLR and CRN reside in gene-sparse, repeat-rich regions, potentially subject 162 to rearrangement and rapid mutation, raising the concept of a ‘two-speed’ genome in evolutionary terms 163 (Haas et al., 2009; Raffaele et al., 2010). Recent years have witnessed intense efforts to determine the 164 biochemical function of RXLR effectors, notably by determining their protein targets in the host. The first 165 target identified was ubiquitin E3 ligase CMPG1, which AVR3a stabilizes to prevent programmed cell death 166 in response to elicitors such as INF1 (Bos et al., 2010). 167

Breeding for late blight resistance was initiated by James Torbitt of Belfast in the 1870s, inspired by 168 the theories of Charles Darwin, with whom he shared considerable correspondence (DeArce, 2008). More 169 than 130 years later, breeding efforts have yet to provide a durable solution. Occasionally, local successes 170 are reported; for example variety C88, widely cultivated in China for over a decade, retains its resistance (Li 171 et al., 2011). Many resistance (R) genes effective against races of P. infestans have been identified 172 (Rodewald and Trognitz, 2013). Only with the recent identification of avirulence genes, all of which encode 173 RXLR effectors, can breeders incorporate knowledge of how P. infestans evades detection into their 174 breeding strategies (Vleeshouwers et al., 2011). Such information is critical to predicting the durability of R 175 genes. Using effectors as tools to rapidly identify R genes is a powerful step towards rational selection and 176 combination of lasting resistance (Vleeshouwers et al., 2008). 177

We still know relatively little about how P. infestans effectors manipulate host plants to establish 178 disease. How do they translocate into host cells? Do they define host range? How do they work in concert to 179 modulate complex networks of regulatory processes occurring in the host? Only recently have researchers 180 started to adopt structural biology to fully investigate functional relationships between interacting pathogen 181 and plant proteins (Wirthmueller et al., 2013). Could such research catalyse the modification of molecular 182 interactions in favor of the plant immune system through synthetic biology approaches? 183

The basic knowledge gained for P. infestans in the last decade is starting to impact management of 184 the late blight disease. For controlling late blight, farmers largely rely on agrochemicals, many with unknown 185 modes-of-action. Emergence of insensitive strains is possible due to high mutation rates in the pathogen 186 (Randall et al., 2014). The phenylamide Metalaxyl was one of the first chemicals that exhibited specificity to 187 oomycetes. Soon after its introduction and widespread use, fully insensitive strains emerged, but only 35 188 years later was the molecular basis of this insensitivity uncovered (Randall et al., 2014). Mining the P. 189 infestans genome has revealed many novel proteins with domain combinations that are unique for 190 oomycetes (Seidl et al., 2011). Several likely function in signalling might be promising fungicide targets. 191 Mode-of-action studies are more accessible thanks to the available genomics resources, transformation 192 tools and marker strains with tags to visualize the cytoskeleton and various subcellular compartments (Ah-193 Fong and Judelson, 2011; Meijer et al., 2014). In resistance breeding the efficacy of stacking R genes into 194 favored cultivars, either by introgression or trans- or cisgenesis, is being investigated (Tan et al., 2010). 195 However, for success, the R genes to be combined should be carefully selected based on our knowledge of 196 A

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variation in the pathogen population, and the attendant vulnerability to resistance being overcome 197 (Vleeshouwers et al., 2011). Future efforts should consider introducing multiple barriers, perhaps combining 198 R proteins with, for instance, membrane-localized pattern recognition receptors or components governing 199 nonhost resistance to P. infestans. Even then it is unlikely that plant resistance can fully control late blight. 200 Agrochemicals should not be abandoned. On the contrary, for more rational fungicide design, novel targets 201 should be identified, an endeavour that will be enhanced by an even more profound insight into the biology 202 of P. infestans. 203 204 2. HYALOPERONOSPORA ARABIDOPSIDIS 205 206 H. arabidopsidis (formerly H. parasitica and Peronospora parasitica), is one of 700 downy mildew species 207 within the Peronosporaceae (Thines, 2014). Downy mildew pathogens cause harmful diseases on many 208 important crops, notably Peronospora and Hyaloperonospora spp. on brassica crops, P. viticola on grape, 209 Peronosclerospora spp on maize and sorghum, Pseudoperonospora cubensis on cucurbits, and Bremia 210 lactucae on lettuce (Lucas et al., 1995). 211

Most downy mildews have narrow host ranges and are completely dependent on their host for 212 growth and reproduction. They can survive in the soil as quiescent oospores that initiate infection through 213 roots. Spread of the pathogen mostly occurs through airborne sporangiospores that are formed on the lower 214 leaf surface, giving downy patches (Fig. 3). These spores germinate on the plant surface and penetrate by 215 forming appressoria (Koch and Slusarenko, 1990). Once past the epidermis, hyphae grow intercellularly 216 and, similar to Phytophthora spp. and other downy mildews, develop haustoria, specialized structures that 217 may function in feeding and suppression of host defense by targeted secretion of effectors (Fig. 4, Whisson 218 et al., 2007) 219

H. arabidopsidis is a prominent pathogen in natural populations of Arabidopsis thaliana (Holub, 220 2008; Coates and Beynon, 2010). As such, it was adopted in the 1980’s as one of two pathogens of 221 Arabidopsis, along with the bacterium Pseudomonas syringae (Koch and Slusarenko, 1990). The top 10 222 ranking of H. arabidopsidis reflects the subsequent success of the Arabidopsis- H. arabidopsidis 223 pathosystem. H. arabidopsidis was initially utilized as a “physiological probe” of the Arabidopsis immune 224 system (Holub et al., 1994). This research led to the cloning of the first plant disease resistance (R) genes 225 against an oomycete, better understanding of the evolutionary dynamics of R genes, definition of broadly 226 important immune system regulators, identification of downy mildew-resistant mutants, and genetic 227 definition of the complexity of the plant immune signalling network (reviewed in Slusarenko and Schlaich, 228 2003, Coates and Beynon, 2010, Lapin & Van den Ackerveken, 2013). On the pathogen side, research is 229 hampered by the lack of protocols for culturing and genetic transformation, established techniques with 230 other oomycetes like P. infestans. However, work in the early 2000s led to the development of genetic maps 231 and DNA libraries that enabled discovery of the first avirulence effector (Allen et al., 2004), and later to the 232 RXLR effector family (Rehmany et al 2005). 233

Genome sequencing of H. arabidopsidis isolate Emoy2, completed in 2010, unveiled 134 predicted 234 RXLR effectors and other components of the H. arabidopsidis secretome (Baxter et al., 2010). Notably, this 235 report also revealed important genomic signatures of obligate biotrophy that have evolved convergently in 236 other obligate oomycete and fungal lineages (reviewed in McDowell, 2011). Protein interaction assays 237 showed that H. arabidopsidis effectors target a highly interconnected host machinery helping to define a 238 representative plant-pathogen interaction network (Mukhtar et al., 2011). In addition, several high 239 throughput functional studies have investigated effectors’ subcellular localizations, suppression of immune 240 responses, molecular targets, and cognate immune receptors (Cabral et al., 2011, Cabral et al., 2012, 241 Caillaud et al., 2011, Fabro et al., 2011). 242

Future studies with the H. arabidopsidis experimental system will include a) direct or 243 Agrobacterium-mediated transformation for genetic manipulation required for the molecular analysis of 244 downy mildew pathogenicity; b) establishment of the temporal hierarchy of effectors during penetration, 245 colonization and sporulation which may serve as a blueprint for a better understanding of the molecular 246 basis of biotrophy; c) revealing the role of genetic recombination and epigenetics on the emergence of new 247 A

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effectors; d) development of tools to understand how plant-originated molecules regulate pathogen 248 response; and e) relevance of interspecies transfer of small RNAs. These investigations on H. arabidopsidis 249 will continue to provide new insights into the molecular mechanisms of downy mildew pathogenicity, and 250 contribute to comparative and functional analysis of (obligate) biotrophic oomycete and fungal pathogens. 251 252 3. PHYTOPHTHORA RAMORUM 253

Phytophthora ramorum is the most destructive disease of oaks worldwide and the cause of sudden oak 254 death, sudden larch death and ramorum blight (Werres et al., 2001; Grünwald et al., 2008; Rizzo et al., 255 2005; Brasier and Webber, 2010) (Fig. 5). The host range of P. ramorum is one of the widest of any 256 Phytophthora spp., and includes many species of hardwood trees and ornamentals. To date, four distinct 257 clonal lineages are recognized by various molecular markers and two mating types are observed (Van 258 Poucke et al., 2012; Grünwald et al., 2009). These lineages appear anciently diverged, yet have emerged 259 repeatedly in both North America and Europe (Grünwald et al., 2012; Goss et al., 2009a) (Fig. 6). Lineages 260 NA1, NA2 and EU1 are currently found in Canada and the US, while EU1 and EU2 exist in Europe (Goss et 261 al., 2009b, 2011; Van Poucke et al., 2012; Grünwald et al., 2012). These lineages differ in aggressiveness 262 in controlled assays, while field experiments have not been conducted given quarantine restrictions 263 (Grünwald et al., 2008; Elliott et al., 2011; Kasuga et al., 2012; Van Poucke et al., 2012; Hüberli et al., 264 2012). Unique ecological attributes such as a wide host range, survival of dry, hot Mediterranean summers, 265 combined with the ability to reproduce from chlamydospores, are thought to provide the basis for sudden 266 oak death in California (Garbelotto and Hayden, 2012). 267

The availability of the genome sequence just a few years after the identification of the pathogen 268 provided rapid and novel insights into the biology of this pathogen (Tyler et al., 2006). Like other 269 Phytophthora spp., P. ramorum has a large number of candidate effectors interacting with the plant host 270 including RXLR, Crinkler and the necrosis and ethylene-inducing peptide 1 (Nep1)-like protein (NLP) gene 271 families (Tyler et al., 2006; Goss et al., 2013). RXLR effectors diversify rapidly despite the clonality of this 272 organism, using mechanisms such as loss or gain of repeated domains, recombination or gene conversion 273 among paralogs, and selection on point mutations (Goss et al., 2013). Recent work focused on the 274 discovery of endogenous small RNAs and description of the silencing machinery (Fahlgren et al., 2013). P. 275 ramorum and two other Phytophthora spp. that were examined produce two primary, distinct 21- and 25-276 nucleotide small RNA classes including a novel microRNA family. Two argonaute classes and two dicer-like 277 proteins appear to be involved in each pathway (Fig. 7), but this remains to be formally tested. Epigenetic 278 mechanisms were recently implicated in phenotypic diversification, namely colony morphology, colony 279 senescence, and virulence on coast live oak and California bay laurel (Kasuga et al., 2012). 280

P. ramorum provides a unique opportunity for studying the evolution of a genome that has two 281 mating types yet appears to lack sexual reproduction in the known field populations. The epigenome, 282 transcriptome and proteome of P. ramorum remain a mystery. The apparent documented phenotypic 283 variation driven by host association might be epigenetic in nature and invites further study. Several basic 284 questions remain unanswered. Why does P. ramorum have such a high number of RXLR effectors given 285 that the genome is adapted to a wide host range? What are the genes conferring a wide host range 286 compared to P. infestans and P. sojae? Is there a center of origin? This center might be located in Asia 287 where the closest relative, P. lateralis has been found in an old growth Chamaecyparis forest (Brasier et al., 288 2010). Discovery of a center of origin might also reveal a host with which P. ramorum might have coevolved 289 providing for a mechanism of R gene discovery, a critical tool needed for managing sudden oak death. 290 Comparative genomics of related species like P. lateralis, P. syringae and P. hibernalis, amongst others will 291 provide novel insights into core effectors and genes under purifying selection in the clade, but diverged 292 among clades. 293 A

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4. PHYTOPHTHORA SOJAE 294 295 Root rot disease of soybean caused by Phytophthora sojae was first recognized in North America in the 296 1950s (Kaufmann & Gerdemann, 1958, Hildebrand, 1959). The alternative names P. megasperma var. 297 sojae and P. megasperma f. sp. glycinea were commonly used for this species in the past (Erwin & Ribeiro, 298 1996). Infection of soybean by P. sojae, which is a hemibiotroph, typically initiates below ground and 299 eventually produces spreading cankers that destroy root tissues and travel up the stem (Fig. 8). The 300 pathogen thrives in wet conditions and in compacted or heavy clay soils. Motile, water-borne zoospores are 301 released from sporangia and are attracted to soybean root exudates (Morris & Ward, 1992). P. sojae is 302 homothallic and creates abundant, thick-walled sexual oospores that are long-lived and provide a soil-borne 303 inoculum. P. sojae has a narrow host range and its economic damage is limited to soybean. It has been 304 suggested that P. sojae originated in North America as a pathogen of lupins as some 26 species of the 305 genus Lupinus are susceptible to infection (Erwin & Ribeiro, 1996). In laboratory tests, P. sojae can also be 306 made to infect lima bean (Phaseolus lunatus), string bean (Phaseolus vulgaris), and cranesbill (Geranium 307 carolinianum) (Hildebrand, 1959, Erwin & Ribeiro, 1996). P. sojae is among the most damaging disease 308 problems that confront soybean growers (Wrather & Koenning, 2006). Management of P. sojae in soybean 309 has primarily relied on breeding for resistance. 310

Research on P. sojae has focused on mechanisms of pathogen virulence, host resistance, and the 311 molecular basis of recognition between P. sojae and soybean (Tyler, 2007, Gijzen & Qutob, 2009, Dorrance 312 & Grünwald, 2009). P. sojae, along with P. ramorum, was the first oomycete to have its genome sequenced 313 (Tyler et al., 2006). Genomic information, including gene expression data and multiple genome sequences, 314 have driven rapid progress (Qutob et al., 2000, Torto-Alalibo et al., 2007, Wang et al., 2011). The genome of 315 P. sojae, and later other oomycetes (Haas et al., 2009), was found to be partitioned into stable regions rich 316 in housekeeping genes displaying extensive synteny with other oomycete genomes, interspersed with 317 dynamic, transposon-rich regions that contain rapidly evolving genes implicated in virulence (Tyler et al., 318 2006, Jiang & Tyler, 2012) (Fig. 9). Multiple large, rapidly diversifying families of genes with functions in 319 virulence were discovered (Tyler et al., 2006, Jiang & Tyler, 2012) including hydrolases, hydrolase inhibitor 320 proteins, toxin-like proteins such as the NLP family (Qutob et al., 2006), and two huge, diverse classes of 321 effector proteins (RXLR and Crinkler effectors) (Tyler et al., 2006, Jiang et al., 2008, Haas et al., 2009) that 322 can cross into the cytoplasm of host plant cells (Dou et al., 2008, Kale et al., 2010) (Fig. 9). All 11 avirulence 323 genes cloned from P. sojae proved to encode RXLR effectors detected by host R proteins (Jiang & Tyler, 324 2012) (Fig. 9). Genetic inheritance studies in P. sojae using molecular markers led to discoveries of high-325 frequency gene conversion (also referred to as loss of heterozygosity) (Chamnanpunt et al., 2001) and 326 epigenetic silencing (Qutob et al., 2013) as important mechanisms underlying pathogen variation. 327

Many areas of future research into P. sojae will be common to other oomycete and fungal 328 pathogens. One will be defining in detail how apoplastic and host cell-entering effectors of P. sojae 329 manipulate soybean physiology to enable infection, including the soybean molecules targeted by the 330 effectors (Fig. 9), and the mechanisms by which RXLR and Crinkler effectors gain entry (Fig. 9). Identifying 331 the P. sojae effectors most important for infection (e.g. using new tools for genetic manipulation such as 332 tailored nucleases) should lead to new targets for disease control. The cell biology of P. sojae infection 333 (Enkerli et al., 1997) must be defined in much more detail, incorporating the spatial distributions of pathogen 334 and host transcripts, proteins and metabolites. Understanding fully the role of epigenetic mechanisms in 335 generating variation in pathogen populations will also be important for designing effective strategies for 336 disease control. Sequencing the genomes and transcriptomes of numerous isolates of P. sojae and its 337 nearest sister species will shed light on the genetic adaptability of the pathogen. In practical terms, a large 338 translational research effort will be required to convert the rapidly accumulating knowledge about the basic 339 biology and pathology of P. sojae and other oomycetes into effective disease control solutions. 340 341 5. PHYTOPHTHORA CAPSICI 342 343 A

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Phytophthora capsici is a highly destructive invasive pathogen that attacks solanaceous (pepper, 344 tomato), legume (lima and snap beans) and most cucurbit hosts (Hausbeck & Lamour, 2004, Leonian, 345 1922). Disease is favored by warm (25 to 28 °C) and wet conditions and the asexual epidemiology is often 346 explosive (Granke et al., 2009). Initial infection is biotrophic followed by transitions within 24 to 48 hours to 347 necrotrophy and the production of deciduous sporangia on the surface of infected tissues (Fig. 10). The 348 importance of the sexual stage differs based on region. In Peru and Argentina and across much of China, 349 long-lived and widely dispersed clonal lineages dominate (Gobena et al., 2012, Hurtado-Gonzales et al., 350 2008, Sun et al., 2008, Hu et al., 2013). This is in contrast to the United States, South Africa and the 351 northern provinces of China where populations have high levels of genotypic diversity and outcrossing is 352 frequent (Gobena et al., 2012, Meitz et al., 2010, Dunn et al., 2010). Once introduced to a field site, P. 353 capsici is difficult to control and often impossible to eradicate. P. capsici grows fast and sporulates heavily 354 on simple media and isolates from sexual field populations are often fecund (Gobena et al., 2012, Lamour & 355 Hausbeck, 2000). P. capsici is one of the most genetically diverse eukaryotic organisms yet described and 356 there is significant genetic variation in the form of single nucleotide polymorphisms within individual 357 genomes and across world populations (Lamour et al., 2012). 358

Current research includes investigation of resistance (host and nonhost) in commercial vegetable 359 and experimental plants, discovery and characterization of genes and proteins driving pathogenicity and 360 virulence, and studies to measure the evolution of natural and laboratory populations. Under controlled 361 conditions, P. capsici infects at least 26 plant families (Granke et al., 2012). Natural resistance to P. capsici 362 in pepper and cucurbits appears to be rare (Mallard et al., 2013) but in tomato, it may be extensive 363 (Quesada-Ocampo & Hausbeck, 2010). Recent molecular studies of the CRN class of effectors indicate 364 they are often localized to the host nucleus and play an important role in the infection process (Chen et al., 365 2013, Stam et al., 2013a, Stam et al., 2013b). Transcriptome studies using RNAseq and microarrays 366 indicate dramatic changes from pre-infective spores through the early biotrophic and later necrotrophic 367 stages of infection (Chen et al., 2013, Jupe et al., 2013). Studies of individual effectors suggest some 368 manipulate the host to allow infection while others trigger plant cell death (Chen et al., 2013, Feng et al., 369 2010, Feng & Li, 2013, Sun et al., 2009). 370

A high quality reference genome and a high density SNP-based genetic linkage map were recently 371 completed (Lamour et al., 2012). These important resources illuminated an important source of asexual 372 genetic variation known as loss of heterozygosity (LOH) (Lamour et al., 2012). LOH occurs when variable 373 length tracts (300bp to >1Mbp) of the diploid genome switch to one of the two possible haplotypes. LOH has 374 been described in laboratory-produced sexual progeny, field isolates maintained on agar media, and field 375 isolates genotyped directly from naturally infected tissue (Hu et al., 2013, Gobena et al., 2010, Hulvey et al., 376 2010). LOH is associated with spontaneous switches from the A2 to the A1 mating type, loss of 377 pathogenicity and reduced virulence (Hu et al., 2013, Lamour et al., 2012). 378

The diversity and plasticity of P. capsici present challenges and unique opportunities for research 379 (Fig. 10). Future research questions include: What is the significance of a highly diverse effector arsenal? 380 Are these effectors important for host-specific interactions and the broad host range? How important is LOH 381 in laboratory and field populations? The genomic plasticity of P. capsici and the ease of laboratory 382 manipulation provide a unique opportunity to measure genome stability and adaptive evolution at a fine 383 scale and may provide useful insights into this and other oomycete pathogens (Lamour & Hu, 2013). 384 385 6. PLASMOPARA VITICOLA 386 387 The obligate biotrophic oomycete P. viticola, causal agent of grape downy mildew, is native to North 388 America and was inadvertently introduced into Europe at the end of the 19th century (Viennot-Bourgin, 1949, 389 Millardet, 1881) causing severe damage to Vitis vinifera, which had evolved in the absence of this pathogen 390 (Galet, 1977, Gessler et al., 2011). Plasmopara viticola belongs to the family Peronosporaceae; its life cycle 391 includes an asexual multiplication phase occurring during the plant vegetative period and a sexual phase 392 that ensures pathogen overwinter survival (Wong et al., 2001). The pathogen overwinters as oospores in 393 dead leaf lesions or as mycelium in infected twigs. In spring, and in particular during rainy periods, oospores 394 A

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germinate to produce sporangia, which are transported by wind or water to the wet leaves near the ground, 395 which they infect through stomata on the lower surface. The mycelium then spreads into the intercellular 396 spaces of the leaf from which sporangiophores arise and emerge through the stoma, ready to start new 397 infection cycles (Gobbin et al., 2005). Downy mildew affects leaves, fruit, and shoots, with young tissues 398 being particularly susceptible to infection (Fig. 11) (Kennelly et al., 2007). A disease cycle may take from 5 399 to 18 days, depending on the temperature, humidity, and varietal susceptibility (Agrios, 2005, Gessler et al., 400 2011). 401

Downy mildew is still most destructive in Europe and in the eastern half of the United States, where 402 it may cause severe epidemics year after year (Agrios, 2005; Madden et al.,1995). When the weather is 403 favorable and no protection against the disease is provided, downy mildew can easily destroy up to 75% of 404 production in a single season (Madden et al., 2000, Rossi & Caffi, 2012). Occasionally, P. viticola can be 405 destructive in other humid parts of the world where V. vinifera is cultivated. 406 In the last fifty years, research on grapevine downy mildew has focused on the pathogen life cycle 407 and epidemiology (reviewed in Gessler et al., 2011), and on the genetic identification of host resistance loci 408 for the establishment of molecular markers (revised in Töpfer et al., 2011). In the last decade, studies on P. 409 viticola have highlighted its enormous potential in developing fungicide resistance (Chen et al., 2007, Blum 410 et al., 2010) and breaking down plant resistance of interspecific hybrids such as ‘Bianca’ (Peressotti et al., 411 2010, Casagrande et al., 2011) and ‘Regent’ (Delmotte et al., 2013). Population genetic studies have been 412 carried out (Fontaine et al., 2013 and references therein) to assess pathogen dynamics and diversity in 413 Europe. Moreover, it was proposed that grapevine downy mildew is not caused by a single species, but 414 instead by a complex of cryptic species that have diverged on Vitaceae (Rouxel et al., 2013). Trade-offs 415 between the size and the number of sporangia produced were reported, leading to ecological advantages 416 for this pathogen (Delmotte et al., 2013). For these reasons the Vitis - P. viticola pathosystem was elected 417 as a prime candidate for studying host specialization of biotrophic plant pathogens (Rouxel et al., 2013) and 418 pathogen adaptation to partial host resistance (Delmotte et al., 2013). 419

Control methods against P. viticola are essentially based on the use of chemical fungicides and 420 application of disease models that serve as decision support systems (Rossi et al., 2013, Vercesi et al., 421 2010, Rossi et al., 2009, Lalancette et al., 1987). Due to the growing public concern on the use of chemical 422 pesticides, and the necessity to adhere to the European Directive 2009/128/EC encompassing a framework 423 “to achieve a sustainable use of pesticides” by promoting Integrated Pest Management (IPM), finding 424 alternative methods for the control of P. viticola on grapevine appears urgent. Several alternative 425 approaches have been proposed in the last 20 years, but none has been transferred to practical use 426 (Gessler et al., 2011). Moreover, the lack of precise characterization of P. viticola isolates is dramatically 427 limiting reliable control of the pathogen. Although molecular studies have previously confirmed a high 428 diversity in the pathogen population, there is a surprising lack of phenotypic characterization of pathotype 429 strains or races which could be used to study the mechanisms of interaction with host genotypes with 430 different levels of resistance (Gómez-Zeledón et al., 2013). In conclusion, interdisciplinary studies and 431 consistent resources should be invested for the study of the P. viticola-V. vinifera pathosystem in order to 432 find new, effective alternatives for the IPM of this highly adaptive and destructive pathogen. 433 434 7. PHYTOPHTHORA CINNAMOMI 435 436 Some call it the ‘biological bulldozer’ for its capacity to destroy natural plant communities across the globe, 437 and disease caused by P. cinnamomi has broad and economically important impacts in forestry and 438 horticulture and in the nursery industry. Like other Phytophthora spp., P. cinnamomi has a number of 439 strategies for survival, propagation and dissemination. The motile zoospore is recognised as the main 440 infective propagule following encystment and attachment to roots and stems (Fig. 12). Recent work 441 suggests that in addition to chlamydospores, P. cinnamomi is also associated with lignituber formation that 442 enables survival under harsh conditions (Jung et al. 2013). Both A1 and A2 mating types are pathogenic but 443 wide differences in distribution and frequency of occurrence suggests the A2 mating type to be more 444 invasive and it is generally recognised as being the more aggressive of the two mating types. Where 445 A

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introduced, this pathogen has had enormous impacts on natural systems including those of Australia, 446 southern Europe and the United States. In Australia, for example, it has been estimated that >3000 largely 447 endemic plant species from numerous plant families are under threat and a new National Threat Abatement 448 Plan has been urgently implemented (Australian Government Department of Environment, 2014) (Fig. 13). 449 Control of disease especially across the large areas of native vegetation affected is still a great problem but 450 some success has been achieved by the use of phosphite in both natural systems and in agriculture 451 (Akinsanmi and Drenth 2013; Crane and Shearer 2013), by the use of calcium amendments to soil (Serrano 452 et al. 2012), and by containment and eradication of spot infections (Dunstan et al. 2010). Climate change is 453 predicted to have a significant influence on the intensity and distribution of disease (Thompson et al. 2014). 454

P. cinnamomi is a challenging organism to work with. Sequencing of its genome has recently been 455 completed (JGI; http://genome.jgi-psf.org/) and has opened up a variety of opportunities to investigate the 456 critical features that enable this pathogen to be so wide-ranging in its hosts. A real change in emphasis in 457 research on P. cinnamomi has occurred in recent times with an increasing number of groups using genomic, 458 transcriptomic and proteomic approaches to understand the pathogen and/or its interaction with the host (for 459 example Reeksting et al. 2014). Experimental plants, including Arabidopsis, Zea mays and Eucalyptus, for 460 which comprehensive genomic information is available, are increasingly used as host-pathogen platforms 461 (for example Allardyce et al. 2013; Dempsey 2012; Rookes et al. 2008). Fundamental studies on P. 462 cinnamomi plant cell wall degrading enzymes and the genes that encode them are being undertaken (Hee 463 et al. 2013; Adrienne Hardham, Australian National University, pers. comm.) as well as examination, at the 464 molecular level, of defense pathways and their control (Eshraghi et al. 2014; Gunning et al. 2013). 465

High throughput, next generation genome sequencing is emerging as an exciting way to examine 466 the pathogen, its hosts and the soil environment and is now being used to examine pathogen ecology and 467 diversity within soils and for analysis of the response of host plants (Treena Burgess, Murdoch University, 468 Australia pers. comm.). Much of our progress in understanding the host-pathogen interactions will come 469 from such approaches. We still don’t know enough about why plants are resistant to this pathogen or what 470 the basis for induced resistance is, for example, following phosphite treatment. The search for host 471 resistance to P. cinnamomi is an active area of research in which signalling pathways and their control are 472 being elucidated. Mapping the disease over the large invaded areas is still not straight forward although 473 advances in remote sensing, high resolution digital photography, hyperspectral imaging (David Guest, 474 Sydney University, Australia pers. comm.) and ‘drone’ technology will likely see our ability in this area to be 475 greatly enhanced in the near future. The era of nanotechnology offers new opportunities for the delivery of 476 molecules that can influence disease outcome yet this area has not been explored to date although 477 preliminary research with various nanoparticle systems (Nadiminti et al. 2013; Hussain et al. 2014) indicates 478 that these may be useful in delivering molecules, including pathogen effectors, DNA and miRNA that may 479 modify host processes and their response to pathogen infection. 480

Future research will focus on the following questions: 1) What are the virulence factors that enable 481 P. cinnamomi to infect and colonise susceptible species and therefore be targeted for control? 2) What 482 constitutes host resistance and how can it be manipulated? Can we look to nanotechnology for some 483 answers? 3) Is phosphite the only chemical that we can use to control P. cinnamomi and how does 484 phosphite alter the host response? 485 486 8. PHYTOPHTHORA PARASITICA 487 488 Phytophthora parasitica (= P. nicotianae) is a worldwide distributed pathogen (Erwin & Ribeiro, 1996). 489 Primarily known to cause tobacco black shank and citrus root rot and gummosis (Fig. 14), it is also 490 responsible for severe foliar and fruit diseases as well as root and crown rots on herbaceous and perennial 491 plant species in more than 250 genera, including solanaceous crops (Fig. 15), and horticultural and fruit 492 trees (Cline et al., 2008). P. parasitica produces both asexual zoospores and thick-walled sexual oospores. 493 Oospores constitute a potential source of genetic variation, and with resting chlamydospores contribute to 494 survival in unfavorable conditions in soil or within infected plant tissues. 495 A

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Contrasting the broad host range observed at the species level, most individual P. parasitica 496 isolates display host preference (Erwin & Ribeiro, 1996). Frequent cases of differential virulence on a range 497 of hosts have been revealed (Colas et al., 1998; Matheron & Matejka, 1990), pointing out the need to 498 decipher the genetic structure of P. parasitica on a global scale. In agreement with pathogenicity tests, 499 recent single nucleotide polymorphism (SNPs) analyses conducted with mitochondrial and nuclear genes 500 revealed a specific association between host of origin and genetic grouping that was particularly evident for 501 tobacco and citrus isolates (Mammella et al., 2011; Mammella et al., 2013). In contrast, no clear genetic 502 structure was revealed for isolates from other hosts, especially potted ornamentals in nurseries. A significant 503 geographic structuring was revealed for tobacco but not for citrus isolates (Bonnet et al., 1994; Colas et al., 504 2001; Mammella et al., 2013). Further studies relying on whole-genome sequencing programs (see below) 505 are necessary to determine if these molecular groups represent evidence of physiological races, pathotypes 506 or even subspecies within P. parasitica. 507

Since some isolates may also infect many hosts, including Arabidopsis thaliana (Attard et al., 2008; 508 Attard et al., 2010), P. parasitica emerges as an ideal pathogen to develop specific studies aiming to 509 advance our knowledge on mechanisms underlying general pathogenicity and those governing host 510 specificity. Furthermore, P. parasitica is phylogenetically related to P. infestans in Phytophthora clade 1, and 511 overlaps its host range, which includes potato (Taylor et al., 2008). Comparative analyses on these two 512 species varying in genome size (83 and 240 Mb for P. parasitica and P. infestans, respectively) will facilitate 513 the understanding of the evolution behind pathogenicity and host range among Phytophthora spp. Through 514 the sequencing of the genome of isolates of diverse host range and geographical origins, the international 515 "Phytophthora parasitica genome initiative" project will enable the characterization of genes that determine 516 host range. In-depth genomic and transcriptomic analyses of 14 sequenced genomes are currently being 517 performed. Special efforts are devoted to characterize the repertoire of effector proteins. As a broad host 518 range pathogen, P. parasitica provides a unique opportunity for intra- and interspecific comparative 519 analyses, looking for the presence of various effector families, their organization, their role in plant 520 recognition and infection, and their evolution among strains and species that display broad or restricted host 521 ranges. The identification of conserved effector groups, as well as of other pathogenicity genes, will allow 522 evaluation of the evolutionary pressures of exposure to different host-defense responses to the 523 diversification of effectors and their role in adaptation to host plants. 524

P. parasitica has not been as heavily studied as its economic importance would warrant. However, 525 it is likely to gain importance in the foreseeable future due the following reasons. First, it is prominent in 526 nurseries of potted ornamentals and fruit tree species, the trade of which seems to represent one of the 527 most efficient dissemination pathways of Phytophthora (Moralejo et al., 2009, Olson & Benson, 2011). This 528 makes P. parasitica an ideal species to study diffusion pathways of Phytophthora and other soilborne 529 pathogens on a global scale. For aesthetic reasons, the ornamental industry requires extensive use of anti-530 oomycete chemicals (Olson et al., 2013). The global nursery trade thus increases the risk of the rapid 531 spread of resistant P. parasitica strains to new areas following an inappropriate use of fungicides and 532 constitutes a growing threat to local agriculture and natural ecosystems (Brasier, 2008). In addition, P. 533 parasitica will likely benefit from the warming climate. Its host range generally includes those of other 534 species of prime economic importance (P. infestans, P. capsici, P. citrophthora) but generally requires 535 warmer conditions than these potential competitors (Erwin & Ribeiro, 1996). Consequently, the foreseen 536 global warming is likely to provide a catalyst for the geographical expansion of this species, as has already 537 been proposed under Mediterranean climates (Andres et al., 2003; Saadoun & Allagui, 2008) and Eastern 538 India (Guha Roy et al., 2009). 539 540 9. PYTHIUM ULTIMUM 541 542 Pythium resides in the peronosporalean lineage of oomycetes, with Phytophthora and downy mildews (Dick, 543 2001). One of its most significant members is Py. ultimum, which causes damping-off and root rot on >300 544 diverse hosts including corn, soybean, wheat, Douglas fir, and ornamentals (Farr and Rossman, 2014). Py. 545 ultimum is a common inhabitant of fields, ponds and streams, and decomposing vegetation worldwide. 546 A

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Contributing to the species' ubiquity is its ability to grow saprotrophically in soil and plant residue, a trait 547 shared by most Pythium spp. but not other peronosporaleans, which must colonize living hosts. Py. ultimum 548 is also homothallic, producing oospores capable of long-term survival (Martin and Loper, 1999). Mycelia 549 and oospores in soil thus can initiate infections of seeds or roots leading to wilting, reduced yield, and 550 mortality (Fig. 16a). Disease management is difficult but focuses on sanitation, fungicides, and biological 551 control, particularly in greenhouses. Quantitative resistance is reported in several hosts but has a limited 552 effect (Lucas and Griffiths, 2004, Wang and Davis, 1997). 553 The role of asexual spores depends on the strain. Py. ultimum is a species complex that includes Py. 554 ultimum var. ultimum and var. sporangiiferum, which have indistinguishable ITS sequences (Schroeder et 555 al., 2013). Sporangia and zoospores are produced rarely by the former, but abundantly by the latter. Py. 556 ultimum var. ultimum makes sporangia-like hyphal swellings which germinate to form infective hyphae (Fig. 557 16b; Stanghellini and Hancock, 1971). Similar to other oomycetes it also produces sexual oospores (Fig. 558 16c). 559 Genome sequencing of several Pythium spp. including Py. ultimum var. ultimum and var. 560 sporangiiferum has provided insight into their biology (Levesque et al., 2010, Adhikari et al., 2013). 561 Annotated in the ca. 43 Mb genomes of two subspecies are 15,290 and 14,086 genes, respectively, which is 562 less than the smallest Phytophthora but more than downy mildews. Whether this difference in gene number 563 is significant requires more analysis, since the two assemblies vary in quality and only single isolates were 564 sequenced. Large size variation between isolates was reported based on electrophoretic karyotyping 565 (Martin, 1995). 566 Evident nevertheless from genome analysis are many differences between Pythium and Phytophthora, 567 including a 25% reduction in Pythium of the fraction of proteins that are secreted. In part, this reflects the 568 absence of RXLR effectors, which may be unneeded for necrotrophic lifestyles. Pythium also lacks 569 haustoria, which biotrophic and hemibiotrophic oomycetes appear to use to deliver RXLRs to host cells 570 (Whisson et al., 2007). Another likely sign of the necrotrophy of Pythium is its greater abundance of lipases 571 and proteases. Pythium however lacks cutinases and pectin esterases, but this is probably because it 572 infects primarily nonsuberized tissue while Phytophthora (which encodes those enzymes) can penetrate 573 plant cuticles. Pythium encodes sufficient enzymes for macerating host cell walls, but cannot degrade some 574 ubiquitous wall components such as xylans, which suggests that soluble sugars may be a more important 575 carbon source (Zerillo et al., 2013). 576 Markers derived from Py. ultimum genomes should help reveal what distinguishes the subspecies and 577 provide more resolution to population studies, which can help identify the sources of outbreaks. Studies of 578 Py. ultimum var. ultimum indicated that populations are not clonal and some outcrossing may occur (Francis 579 and St. Clair, 1997). A bigger evolutionary question concerns the organization of the Pythium genus as a 580 whole. Division of the >120 Pythium species over five new genera was proposed based on sequence 581 analysis of two loci (Uzuhashi et al., 2010). If accepted by the community, Py. ultimum would be renamed 582 Globisporangium ultimum. 583 Of much interest is the interaction between Py. ultimum and biocontrol agents such as Trichoderma, 584 Streptomyces, Pythium oligandrum (a mycoparasitic member of the genus), and others (Gracia-Garza et al., 585 2003, Martin and Loper, 1999). The extent to which these organisms function by altering rhizosphere 586 microflora or attacking Py. ultimum directly is an open question (Naseby et al., 2000, Vallance et al., 2012); 587 Py. ultimum competes poorly with organisms having higher saprotrophic capabilities. 588 Py. ultimum is just one of many destructive members of the genus, with Py. aphanidermatum and Py. 589 irregulare also topping lists of important pathogens (Martin and Loper, 1999). Opportunities for 590 understanding their biology, ecology, and evolution have been enhanced by genome sequencing. DNA-591 mediated transformation was achieved for several species, but few studies of gene function are reported 592 (Weiland, 2003, Grenville-Briggs et al., 2013). Since core promoter structure appears conserved within the 593 Peronosporales, vectors and technologies applied to species such as Phytophthora should be transferable 594 to Pythium (Roy et al., 2013). Pythium spp. are easily grown and have the potential to develop into valuable 595 experimental systems for necrotrophic and saprotrophic oomycete lifestyles. Their growth on byproducts of 596 the agro-food industry has also attracted interest as sources of polyunsaturated fatty acids for human 597 A

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consumption (Stredansky et al., 2000). 598 599 10. ALBUGO CANDIDA 600 601 White blister rust is a disease caused in many dicotyledonous plant species by obligate biotrophic parasites. 602 For example, A. candida infection of Brassica juncea (Indian mustard) can result in significant crop losses in 603 India (Awasthi et al., 2012), Canada (Rimmer et al., 2000) and Australia (Kaur et al., 2008). The white rusts, 604 order Albuginales, are phylogenetically distant from the Peronosporales and probably represent an 605 independent acquisition of biotrophy (Thines and Spring, 2005; Thines and Kamoun 2010). All Albugo 606 species infecting the Brassicaceae were thought to be races of A. candida, but molecular studies of isolates 607 from various hosts and locations led to the description of specialists, for example Albugo laibachii on 608 Arabidopsis thaliana (Thines et al., 2009; Thines 2014). Specific A. candida races can grow on diverse plant 609 hosts, including Brassicaceae, Cleomaceae and Capparaceae (Thines, 2014). Albugo spp. provide an 610 interesting experimental system for the study of plant immune suppression, disease resistance and host-611 pathogen co-evolution. 612

Albugo spp. reproduce asexually via zoosporangia, which release flagellated motile zoospores 613 upon incubation in water. On the surface of a plant leaf, zoospores settle in stomata, and each extends a 614 germ tube into the sub-stomatal chamber (Holub et al., 1995). Coenocytic hyphae then grow intercellularly 615 through the plant. Small globose haustoria penetrate into plant cells (Soylu et al., 2003). When an Albugo 616 infection is mature, zoosporangia rupture the plant epidermis with force and enzymatic digestion (Heller and 617 Thines, 2009). This results in characteristic “white blister” pustules. Albugo also has a sexual cycle, 618 producing tough oospores that can survive difficult environmental conditions (Petrie, 1975). During systemic 619 infection of Brassicacea hosts, the inflorescences become misshapen, forming so-called ‘stagheads’ (Fig. 620 17A). 621

Albugo infection has long been associated with “green islands” where infected tissue appears 622 healthy and senescence is delayed. Infection by Albugo also greatly enhances susceptibility to co-infections 623 with downy mildews (Bains and Jhooty, 1985; Crute et al., 1994). Cooper et al (2008) investigated the ability 624 of A. laibachii and A. candida to suppress host immunity. They showed that A. laibachii can suppress the 625 “runaway cell death” of Arabidopsis lsd1 mutants after inoculation with avirulent H. arabidopsidis. 626 Furthermore, when pre-infected with virulent A. laibachii, resistant Arabidopsis accessions were no longer 627 resistant to avirulent H. arabidopsidis isolates (Fig. 17B), lettuce downy mildew or powdery mildew. 628 Suppression was also observed on B. juncea with A. candida and Brassica downy mildew (Cooper et al., 629 2008). These results suggest that Albugo is effective at broad suppression of plant immunity, including 630 effector-triggered-immunity. 631

The first step to understanding how Albugo spp. impose such susceptibility is to examine their 632 genomes. Links et al (2011) and Kemen et al (2011) sequenced A. candida and A. laibachii genomes, 633 respectively. The genomes are around 40 Mb and compact; about 50% of the assemblies consist of coding 634 sequences (see Fig. 18). Both genomes show adaptions to obligate biotrophy; they are missing sulfite 635 oxidases, nitrate and nitrite reductases and in the case of A. laibachii the whole molybdopterin biosynthesis 636 pathway. The A. candida secretome consists of 929 proteins (without transmembrane domains) compared 637 to 672 in A. laibachii, perhaps reflecting its wider host range. Within the secretomes there is no enrichment 638 of putative RXLR effectors. Kemen et al (2011) discovered the CHXC (cysteine, histidine, any amino acid, 639 cysteine) motif at the N-terminus of a class of candidate effectors. The CHXC-containing N-terminus is 640 sufficient to translocate the C-terminus of P. infestans AVR3a (an RXLR effector) into host cells (Kemen et 641 al., 2011). 642

Several A. candida races can infect some but not all A. thaliana accessions and from crosses 643 between resistant and susceptible accessions, an R gene against four A. candida races, WRR4 (encoding a 644 TIR-NB-LRR R-protein), was identified (Borhan et al., 2008). WRR4 can also provide resistance to A. 645 candida when transformed into susceptible cultivars of B. napus and B. juncea (Borhan et al., 2010). In A. 646 thaliana, RAC1 (also encoding a TIR-NB-LRR R-protein) confers resistance to A. laibachii (Borhan et al., 647 2004). The inheritance of avirulence of a B. juncea isolate (Ac2V) was studied through a cross between two 648 A

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A. candida isolates; this work predicted a single avirulence gene for the incompatibility between Ac2V and 649 B. rapa (Adhikari et al., 2003). 650

There are open questions about Albugo from both fundamental and translational perspectives. 651 Thines (2014) speculated that the broad host range of the A. candida meta-population is maintained through 652 frequent genetic exchange where the host range of individual isolates overlap. Comparing the genomes of 653 multiple isolates from different hosts would test this hypothesis and build up a clear picture of population 654 variation. This would also aid the discovery of new effector candidates through the identification of secreted 655 proteins under strong selective pressure. A more extensive analysis of Albugo effectors should be made. 656 The presence of the CHXC effectors inside host cells needs to be confirmed and the translocation 657 mechanism elucidated. It is unclear which Albugo effector proteins are recognised by the few known R-658 proteins. A. thaliana cannot be colonised by most A. candida isolates. The molecular basis for this 659 resistance could be exploited to introduce durable resistance to Brassica crops. 660

Lastly and perhaps most interestingly: how does the remarkable defense suppression by Albugo 661 work? We need to define the extent of this suppression- what other pests or pathogens can grow on plants 662 infected with Albugo? What changes occur in the microbiome of Albugo-infected leaves in the field? Recent 663 data suggested that Albugo could be widespread as an asymptomatic endophyte (Ploch and Thines, 2011). 664 The implications of these infections as a reservoir for i) Albugo and ii) further Albugo suppression-enabled 665 infections by other species remain to be discovered. 666 667 CONCLUSION 668 669 Although the study of oomycete plant pathogens has always been an important topic in plant pathology, it 670 has recently taken an even more central role with the advent of genomics and the discovery of large effector 671 repertoires in oomycete genomes. This article provides a benchmark for future trends. Many topics remain 672 to be investigated in more depth. For example, the large number of pathogenic species and the wide 673 diversity of host species and host ranges should provide a rich basis for investigating the genetic and 674 physiological basis for host adaptation and specialization. It will be also be interesting to track how the Top 675 10 list will develop in the coming years. Many pathogenic oomycetes among the 33 listed (Table 2) have 676 evolved unique adaptations in their parasitic lifestyle that will undoubtedly reveal fascinating processes and 677 mechanisms. Thus future research efforts should take into account a diverse spectrum of taxa beyond the 678 widely studied species. 679 680 ACKNOWLEDGEMENTS 681

The authors would like to thank Dr. Diane Hird for assistance with several aspects of the project. 682 Sophien Kamoun, Oliver J. Furzer and Jonathan D.G. Jones received funding from the European Research 683 Council (ERC), the UK Biotechnology and Biological Sciences Research Council (BBSRC), and the Gatsby 684 Charitable Foundation. Ronaldo Dalio thanks the National Council for Scientific and Technological 685 Development (CNPq/CsF Brazil - 313139/2013-0) for financial support. Michelina Ruocco receives support 686 from Conoscenze Integrate per Sostenibilità ed Innovazione del Made in Italy Agroalimentare (CISIA –687 MIUR). Leonardo Schena was supported by grant FIRB 2010 - RBFR10PZ4N from the Italian Ministry of 688 Education, University and Research (MIUR). Andreia Figueiredo receives support from the Portuguese 689 Foundation for Science and Technology (Grant nº SFRH / BPD / 63641 / 2009). Xiao-Ren Chen was 690 financed by National Natural Science Foundation of China (Grant no. 31101395) and Jiangsu Province 691 Basic Research Program (Natural Science Foundation) of China (Grant no. BK2011443). Howard S. 692 Judelson and Brett M. Tyler were supported by grants 2011-68004-30154 and 2011-68004-30104, 693 respectively, from the Agriculture and Food Research Institute of the National Institute of Food and 694 Agriculture of the USDA. Mark Gijzen was supported by Agriculture and Agri-Food Canada GRDI program. 695 Niklaus J. Grünwald was supported by grant 2011-68004-30154 from the USDA National Institute of Food 696 and Agriculture, the National Research Initiative Competitive Grants Program grant 2008-35600-18780, the 697 USDA Agricultural Research Service CRIS 5358-22000-039-00D, the Northwest Center for Nursery Crop 698 A

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Research, the US Forest Service, the USDA ARS Floriculture Nursery Initiative and Oregon Department of 699 Agriculture/ Oregon Association of Nurseries. Daniel F.A.Tomé is funded by the UK Biotechnology and 700 Biological Sciences Research Council grant BB/G015066/1. John McDowell is supported by the U.S. 701 Department of Agriculture–Agriculture and Food Research Initiative (2009-03008 and 2011-68004), the 702 National Science Foundation (ABI-1146819), and the Virginia Tech Institute for Critical Technology and 703 Applied Sciences. Fouad Daayf received funding from NSERC, MB-ARDI, McCain Foods, KPPA, and Peak 704 of the Market. Harold J.G. Meijer is funded by The Dutch Technology Foundation STW-NWO (VIDI grant 705 10281). Benjamin Petre is supported by an INRA Contrat Jeune Scientifique and has received the 706 support of the European Union, in the framework of the Marie-Curie FP7 COFUND People 707 Programme, through the award of an AgreenSkills’ fellowship (under grant agreement n° 708 267196). Francine Govers receives support from the Food for Thought program, Wageningen University 709 Fund. 710 711 712 AUTHOR CONTRIBUTIONS 713 714 All authors voted and contributed to writing the 10 sections. SK oversaw the voting and writing. Other 715 authors are listed in reverse order based on the species ranking. The species coordinator(s) are listed last in 716 their section and are preceded by other contributors listed in alphabetical order. The species coordinators 717 are: A. candida (OF and JDGJ), Py. ultimum (HJ), P. parasitica (FP), P. cinnamomi (DC), Pl. viticola (MR 718 and AF), P. capsici (KL), P. sojae (MG and BMT), P. ramorum (NJG), H. arabidopsidis (GvdA and JMcD), 719 and P. infestans (PRJB and FG). 720 721 REFERENCES 722

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FIGURE LEGENDS 1351

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Figure 1. Potato plants with typical late blight lesions. Infection starts when a spore lands on the leaf and 1353 germinates. The germ tube forms an appressorium and an emerging penetration peg pushes into an 1354 epidermal cell. Then the inner cell layers are colonized. During the biotrophic phase, hyphae grow in the 1355 intercellular space while haustoria enter plant cell cavities and invaginate host cell plasma membrane. Later, 1356 P. infestans switches to necrotrophic growth resulting in the death of plant cells and the appearance of 1357 necrotic lesions on the infected tissues. In this phase, hyphae escape through the stomata and produce 1358 numerous asexual spores named sporangia that easily detach and disperse by wind or water. A sporangium 1359 that finds a new host can either germinate directly and initiate a new cycle or, at lower temperatures, 1360 undergo cleavage resulting in a zoosporangium from which 6-8 flagellated spores are released. These 1361 zoospores can swim for several hours but once they touch a solid surface they encyst and germinate to 1362 initiate new infections. Under favourable conditions the pathogen can complete the cycle from infection to 1363 sporulation in 4 days. In the field this cycle is repeated multiple times during one growing season resulting in 1364 billions of spores and a continuous increase of disease pressure. Besides leaves, stems and tubers also get 1365 infected and P. infestans can continue to flourish on the decaying plant material. If not managed properly, 1366 infected seed potatoes or waste on refuse piles are often the sources of inoculum for new infections in 1367 spring. An alternative route for surviving the winter is via oospores, sexual spores that can survive in soil for 1368 many years. P. infestans is heterothallic; isolates are either A1 or A2 mating type and sex organs only 1369 develop when isolates of opposite mating type sense the sex hormone produced by the mate. 1370 1371 Figure 2. One of the many Great Famine memorials around the world. These sculptures on Customs House 1372 Quays in Dublin, by artist Rowan Gillespie, stand as if walking towards the emigration ships on the Dublin 1373 Quayside (courtesy of Michael Seidl). 1374 1375 Figure 3. Hyaloperonospora arabidopsidis disease symptoms on a two-week old Arabidopsis seedling. 1376 Mature sporangiophores are visible as white structures on the right side of the leaf. 1377 1378 Figure 4. Diagram depicting a compatible interaction between H. arabidopsidis and Arabidopsis initiated by 1379 sporangiospore landing on the leaf surface. (S) sporangiospore; (A) appressorium; (N) nucleus; (Hy) 1380 hyphae; (Ha) haustorium; (C) cuticle; (UE) upper epidermis; (PM) palisade mesophyll cells; (SM) spongy 1381 mesophyll cells; (LE) lower epidermis; (Sp) mature sporangiophore. Note: Sporangiophore not drawn to 1382 scale. 1383 1384 Figure 5. Impact of sudden oak death in California. Tanoak mortality evidenced by defoliated or wilted 1385 canopies on the Bolinas Ridge at Mt. Tamalpais, Marin County, California. Photo courtesy of Janet Klein 1386 (Marin Municipal Open Space District). 1387 1388 Figure 6. Inferred pattern of migration of the four clonal lineages of Phytophthora ramorum. Modified from 1389 Grünwald et al. (2012). 1390 1391 Figure 7. Silencing machinery in Phytophthora. (A). Phylogenetic placement of dicer-like (DCL) and (B). 1392 argonaute (Ago) proteins in the genus Phytophthora. See Fahlgren et al. (2013) for more details. Species 1393 correspond to: Arabidopsis thaliana, Paramecium tetraurelia, Phytophthora infestans, Phytophthora 1394 ramorum, Phytophthora sojae, Tetrahymena thermophila, and Toxoplasma gondii. 1395 1396 Figure 8. Phytophthora sojae. A, Diseased soybean plants in the field, infected with P. sojae. Plant height is 1397 20 to 30 cm. B, Susceptible (left) and resistant (right) soybean plants inoculated in the stem with P. sojae, 1398 seven days after infection, illustrating R-gene mediated resistance. Pots are 10 cm in diameter. C, 1399 Germinating oospore of P. sojae growing on water agar. Oospore is 35 µm in diameter. D, Germinating P. 1400 A

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sojae cysts growing on water agar. Cysts are 15 µM in diameter. E, Etiolated soybean hypocotyls 1401 inoculated with a 10 µL water droplet containing 103 zoospores from a virulent (upper) or avirulent (lower) 1402 strain of P. sojae, 48 h after infection, illustrating strain specific variation in avirulence effectors and the 1403 hypersensitive response. Soybean hypocotyls are 5 mm in diameter. 1404 1405 Figure 9. Large diverse families of virulence proteins encoded by the P. sojae genome. (A) P. sojae 1406 genome contains clusters of conserved housekeeping genes (brown) that have conserved orders among 1407 Phytophthora species, separated by dynamic transposon-rich regions that contain genes (red) encoding 1408 virulence proteins, many of which are secreted. (B) Secreted virulence proteins (effectors) may act in the 1409 apoplast, or be transported inside the cell. Cell-entering effectors may have targets in the nucleus or 1410 cytoplasm, and may be detected by resistance proteins (Rps; resistance against P. sojae). 1411 1412 Figure 10. Heavy sporulation and spontaneous morphological variation in the vegetable pathogen P. 1413 capsici. (A) Naturally infected tomato fruit with sporangium production on the surface of the fruit and (B) a 1414 single-zoospore derived field isolate of P. capsici sectoring on V8 agar media following long term storage. 1415 1416

Figure 11. Downy mildew symptoms with well evident sporangiophores on the lower side of a grape leaf 1417 (A) and a young cluster (B). 1418

1419 Figure 12. Cryo-SEM micrograph of P. cinnamomi cyst between two epidermal cells on a root of tobacco. 1420 Note the adhesive material that surrounds the cyst that has been expelled by zoospore peripheral vesicles. 1421 Photo courtesy of Adrienne Hardham, Australian National University. 1422 1423 Figure 13. (A) Individual plants of Xanthorrheoa australis (austral grass tree), a highly suscpetible native 1424 Australian species, infected by Phytophthora cinnamomi within a dry sclerophyll eucalypt forest at Anglesea, 1425 Victoria, Australia. Note the dead and dying plants that have brown, collapsed leaves compared with the 1426 healthy green and erect leaves of plants which are yet to be killed. These individual plants range in age from 1427 approximately 20 years (the smallest in the centre) to around 70 years (green individual on the right of the 1428 image). (B) Advancing disease front caused by invasion by P. cinnamomi in Xanthorrhoea australis- 1429 dominated understorey in eucalyptus open forest at Wilsons Promontory, Victoria, Australia. The disease 1430 has moved from the foregound of the picture, where all susceptible vegetation including X. australis has 1431 been killed and its progress can be seen as a line of dead and dying X. australis (brown collapsed plants) at 1432 the disease margin. Healthy green plants behind them will soon be killed. Loss of the major understorey 1433 components, as in the forground, results in complete structural change and loss of all susceptible species. 1434 1435 Figure 14. Impact of Phytophthora parasitica-infection on citrus plants. A and B: five year-old citrus plants 1436 non-infected and infected with P. parasitica respectively; C and D: symptoms of P. parasitica on stems; C: 1437 non-infected, D: infected plant displaying gummosis symptoms; E and F: leaves and fruits of infected (left) 1438 and healthy (right) plants; G and H: scanning electron microscope images of citrus fine roots infected with P. 1439 parasitica. Yellow arrows show encysted zoospores and germ tube of P. parasitica. Bar represents 20 µm. 1440 A- F: photograph courtesy R. J. D. Dalio; G and H: photographs courtesy of M. E. Escanferla. 1441 1442 Figure 15. Severe infection of Brinjal fruit with P. parasitica. Typical symptoms are brown, soft water soaked 1443 patches which rapidly cover the whole fruit. Brinjal is also known as eggplant (Solanum melongena). 1444 Photograph courtesy of S. Guha Roy. 1445 1446 Figure 16. Pythium ultimum var. ultimum. (A) Pre-emergence damping-off in okra, resulting in the death of 1447 most plants in the front of the row. In the back, disease was controlled using metalaxyl. (B) Terminal 1448 hyphal swellings, bar = 10 µm. (C) Oospore within oogonium, bar = 10 µm. Images adapted from Kida et 1449 al. (2006) with permission. 1450 A

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1451 Figure 17. Disease symptoms of Albugo infections. (A) Disease resulting from the infection of Brassica 1452 juncea with an unknown Albugo species. The misshapen inflorescence phenotype is known as a 1453 “staghead”. (B) An example of immunity suppression by Albugo laibachii: A. thaliana Col-0 is resistant to H. 1454 arabidopsidis Emoy2 via RPP1, but when pre-infected with Al can support the growth of both pathogens. 1455 1456 Figure 18. Albugo spp. have compact genomes. Synteny between A. laibachii, Py. ultimum, H. 1457 arabidopsidis, and P. infestans. The region shown is an example of the relatively dense clustering of genes 1458 in Albugo species. With increasing genome size the distance between both genes increases and re-1459 organisations occur (red, synteny without inversion; blue, inverted regions). Reproduced from Kemen et al, 1460 (2011). 1461

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Table 1. Top 10 oomycetes in molecular plant pathology. 1463 1464 Rank species Common disease

name(s) Number of papers (2005-2014)

Number of votes

1 Phytophthora infestans Late blight 1230 51 =2 Hyaloperonospora

arabidopsidis Downy mildew 137 25

=2 Phytophthora ramorum Sudden oak death, Ramorum disease

378 25

4 Phytophthora sojae Stem and root rot 276 22 5 Phytophthora capsici Blight; stem and fruit

rot; various others 541 17

6 Plasmopara viticola Downy mildew 326 15 7 Phytophthora cinnamomi Root rot; dieback 315 13 =8 Phytophthora parasitica Root and stem rot;

various others 142 10

=8 Pythium ultimum Damping off; root rot 319 10 10 Albugo candida White rust 65 9

1465 The “=” sign before the ranking indicates that the species tied for that position. The number of papers 1466 published in 2005-2014 is based on searches of the Scopus database (http://www.scopus.com) using the 1467 species names as a query. For H. arabidopsidis, a search for the alternative name “Peronospora parasitica” 1468 was also performed and the combined number is shown. Searches with the terms “oomycete*” and 1469 “Phytophthora” yielded 2068 and 4059 articles, respectively. 1470 1471 1472

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1474 Table 2. Other oomycetes species that received votes. 1475 1476 Rank species

11 Aphanomyces euteiches 12 Albugo laibachii 13 Bremia lactucae 14 Phytophthora palmivora 15 Pseudoperonospora cubensis 16 Plasmopara halstedii 17 Peronophythora litchi 18 Peronosclerospora sorghi 19 Peronospora belbahrii 20 Phytophthora alni 21 Phytophthora brassicae 22 Phytophthora cactorum 23 Phytophthora meadii 24 Phytophthora phaseoli 25 Phytophthora plurivora (formerly P. citricola) 26 Plasmopara obducens 27 Pythium aphanidermatum 28 Pythium oligandrum 29 Sclerophthora rayssiae 30 Hyaloperonospora brassicae NR Saprolegnia parasitica (fish parasite) NR Lagenidium giganteum (mosquito parasite) NR Pythium insidiosum (mammalian parasite)

1477 NR, not ranked because the species is not associated to plants. 1478 1479

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Documents

The Top 10 oomycete pathogens in molecular plant pathology